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Inoculation of somatic embryos of sweet potato with an arbuscular mycorrhizal fungus improves embryo survival and plantlet formation
CARVALHO, C.H.S. 2000
Wellington Bressan, Carlos Henrique Siqueira de Carvalho, and David M. Sylvia
Abstract: Responses of somatic embryos of sweet potato (lpomoea batata (L.) Poir., cv. White Star) at different developmental stages to in vitro inoculation with Glomus etunicatum (Becker and Gerdemann) (isolate INVAM FL329) were evaluated. Somatic embryos were grown in glass tubes containing sterilized vermiculite and sand. A layer of natrosol plus White's medium was used as a carrier for arbuscular mycorrhizal (AM) fungal spores. Survival of embryos inoculated with AM fungi was significantly (P < 0.05) greater than that of noninoculated embryos at the rootedcotyledonary-torpedo and rooted-elongated-torpedo developmental stages. Mycorrhizae significantly (P < 0.05) increased plantlet formation only when inoculation occurred at the rooted-elongated-torpedo developmental stage. The growth stage at which the embryos were inserted into the glass tubes exerted a significant influence upon plantlet formation, and plantlet formation was further enhanced by inoculation with G. etunicatum. Plantlet formation was greatest at the rooted-elongated-torpedo stage. These results demonstrate that inoculation of somatic embryos with AM fungi improves embryo survival and plantlet formation, and could enhance use of somatic embryos as synthetic seeds. Key words: mycorrhiza,
Glomus etunicatum,
somatic embryos.
Résumé : Nous avons étudié Ia réponse d'embryons somatiques de Ia pomme de terre sucrée (lpomoea batata (L.) Poir., cv. White Star) à différentes étapes du développement suite à l'inoculation in vitro avec du Glomus etunicatum (Becker et Gerdemann) (isolat INVAM FL329). Les embryons ont été cultivés dans des éprouvettes contenant du sable et de Ia vermiculite stériles. Un enrobage avec du milieu White + Natrosol a servi à l'inoculation de spores du charnpignon des mycorhizes des arbuscules (AM). La survie des embryons inoculés avec le champignon AM a été significativement plus élevée (P < 0.05) que celIe d'embryons non inoculés aux stades de développement des cotylédons qui s'enracinent ou de croissance des racines allongées en processus d'enracinement continuo Les mycorhizes ont significativement favorisé Ia formation de plantules mais seulement lorsque l'inoculation avait lieu au stade de Ia formation des racines allongées qui s'enracinent. Lors de Ia mise en éprouvettes, il a été constaté que le stade de croissance avait une influence significative sur Ia formation de plantules et que celle-ci était favorisée par l'inoculation avec le G. etunicatum. Le stade de développement des racines alIongées était celui qui permettait Ia meilleure production de plantules. Les résultats obtenus confirmem que l'inoculation dernbryons somatiques avec des champignons AM augmente Ia survie des embryons et Ia formation des plantules et quil faudrait favoriser l'utilisation des embryons somatiques comme semences synthétiques. Mots clés : mycorhizes,
Glomus etunicatum,
embryons
somatiques.
[Traduit par Ia Rédaction]
"-- The use of somatic embryos as "synthetic seeds" has potential applications in providing large-scale planting of outstanding genotypes of self-incornpatible species that are difficult to propagate vegetatively, and for propagation of
Received December 20, 1999. Revision received April 5, 2000. Accepted April 6, 2000. Published on NRC Research Press web site July 17,2000.
W. Bressan! and C.H. Siqueira de Carvalho.
EMBRAPA / Embrapa Milho e Sorgo, Departrnent of Soil Microbiology, P.O. Box 151,35701-970, Sete Lagoas, MG, Brazil. D.M. Sylvia. Soil and Water Science Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611-0290, U.S.A. I
Author to whom all correspondence (e-rnail:
[email protected]).
Cano J. Microbiol.
46: 741-743
(2000)
should be addressed
nove I genotypes produced by genetic engineering that are not meiotically stable, and for maintenance of parental inbred lines (Gray et aI. 1987). Germination in the soil requires the somatic embryos to be self-sufficient for root and shoot formation. Taylor (1986) suggested the use of gel material with a supply of nutrients for improved gerrnination and early seedling growth of zygotic seeded species. Schultheis et aI. (1988) reported that macronutrients added to the gel increased the percentage of embryos rooted, and that macronutrients and micronutrients are needed for optimum plant production. Beyond this, beneficial microorganisms like arbuscular mycorrhizal (AM) fungi can be added to the gel material. Mycorrhizal studies have been performed in vitro using axenic plantlets grown frorn seed, and fungi grown in pure culture (Molina 1981; Blal et ai. 1990). Strullu and Romand (1987) reported that plants raised from coated somatic embryos were able to form in vitro typical © 2000 NRC Carrada
Cano
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J. Microbial. VaI. 46, 2000
Table 1. Survival of inoculated and noninoculated sweet potato somatic ernbryos after 30 days of growth in sterilized vermiculite and sand + Natrosol (2% solution) and White's medium. Results are means of 30 replicates. Embryo survival (%)* Embryo type
Noninoculated
Inoculated
Cotyledonary torpedo Rooted-cotyledonary-torpedo Elongated torpedo Rooted-elongated-torpedo
0.0 16.5 2.0 20.8
Da
0.0 Da 22.4 Ba
Bb Ca Ab
with Glomus etunicatum
2.8 Ca 36.9 Aa
*Means within a column followed by the same capital letter and within a row followed by the same lower case letter do not differ significantly at P < 0.05 by Duncari's multiple range test. Significance deterrnined after are sine transformation.
Table 2. Mean plantlet formation and plantlet root colonization Glomus etunicatum (INVAM FL329). Plantlet
formation
of sweet potato somatic embryos
Noninoculated
lnoculated
Cotyledonary
0.0 2.0 3.8 8.2
0.0 Da 2.5 Ca 4.2.Ba 15.6 Aa
Ca Ba Ba Ab
or uninoculated
with
(%)*
Embryo stage torpedo Rooted-cotyledonary torpedo Elongated torpedo Rooted-elongated torpedo
inoculated
with Glomus etunicatum
Plantlet root colonization 0.0 O
8.6 C 15.2 B 28.8 A
*Means within a column followed by the sarne capital letter and within a row followed by the same lower case letter do not differ significantly P < 0.05 by Duncans multiple range test. Significance deterrnined after are sine transformation.
vesicular-arbuscular mycorrhizas when inoculated with vegetative mycelium of Glomus sp. regenerated from sterilized root segrnents. The objective of this study was to investigate the effect of mycorrhizal symbiosis on the development of somatic embryos of sweet potato. The experiment was conducted at the Soil and Water Seience Department, University of Florida. Rooted and unrooted sweet potato (lpomoea batata (L) Poir.) (cv. White Star), somatic embryos of cotyledonary-torpedo and elongated-torpedo shapes, were inoculated with or without disinfected germinated spores of Glomus etunicatum etunicatum (Becker and Gerdemann) (INVAM FL329) to determine effects on embryo survival, plantlet production, and mycorrhizal root colonization. Friable, embryogenic calli from Murashige and Skoog (1962) medium containing 1.0 ~ of 6-benzylaminopurine were removed from plates and collected in beakers. Cell aggregates were gently broken apart using a glass slide, then separated into 335 and 71 O um size fractions using steel sieves, The largest fraction was plated for embryo production in petri dishes (100 x 15 mm) containing Murashige and Skoog (1962) solid medium lacking growth regulators. Plates were incubated in the dark for one week, followed by a 10/14 hour lighrJdark cycle (252 ~01·m-2.Çl) at 26 ± 2°C for 20 days, until normal embryos were obtained. Abnormal embryos, those fused with others or with rnisshapen scutellar tissues, were discarded. Embryos selected at cotyledonary, rooted-cotyledonary, elongated-torpedo, and rooted-elongatedtorpedo stages were placed into a layer of 2% solution of Natrosol (Hung et a!. 1991), amended with White's (1963) medium and inoculated with 50 disinfested AM fungus spores in glass tubes (150 x 25 mm) filled with sterilized vermiculite and sand. Spores were disinfested with a 10% bleach solution with I drop of Tween 20 for 5 min, and washed tive times with sterilized water, There were 30 replicates of each treatrnent arranged in a completely randomized
(%)
at
designo Embryo survival (%), plantlet formation, and mycorrhizal root colonization (%) were evaluated 30 days after the embryos were placed in the glass tubes. The roots were separated from the embryos, and plantlets were gently washed to remove vermiculite and sand particles, and then transferred to tissue-holding capsules. The roots were stained according to Phillips and Hayman (1970) and mycorrhizal root colonization was determined using the grid-line-intersect method (Giovanetti and Mosse 1980). Data were analized using the general linear model (GLM) procedure of SAS (SAS Institute Inc. 1985). Significance of treatments were evaluated by Duncan's multiple range test (P < 0.05). Percentage embryo survival and root colonization data were are sine transforrned prior to analysis. Typical AM symbiosis was found on inoculated plantlets derived from somatic embryos, and no contamination was observed among the inoculated embryos or plantlets. The stage of development of the somatic embryos also affected embryo survival and mycorrhizal syrnbiosis. Mycorrhizal fungus colonization significantly (P < 0.05) improved embryo survival at the rooted-cotyledonary-torpedo and rooted-elongated-torpedo developmental stages (Table I). These responses are likely due to greater availability of nutrients to the somatic embryos due to uptake of nutrients from the media through AM fungal hyphae. Significant differences (P < O.OS} in embryo survival between inoculated and noninoculated somatic embryos occurred when inoculation took place at more advanced developmental stages. Rootedelongated-torpedo embryos had the greatest survival, which indicates that these embryos are better able to adapt to new environmental conditions. Inoculation with G. etunicatum significantly enhanced plantlet formation from somatic embryos at the rooted-elongated-torpedo stage (Table 2). Embryos transferred to the glass tubes at the most advanced stage of maturation had the greatest plantlet formation. The presence of roots in cotyledonary-torpedo embryos did not © 2000 NRC Canada
Notes
enhance plantlet forrnation relative to that of the elongatedtorpedo embryos, a more advanced stage of embryo maturation. This confinns that the stage of embryo maturation is more important than the presence of raots to promote plantlet forrnation, although the presence of roots could improve nutrient uptake fram the media and lead to greater embryo development and plantlet fonnation. The percentage of plantlet roots colonized by mycorrhizal fungi ranged from 8.6% to 28.8% (Table 2). The highest colonization was with rooted-elongated-torpedo embryos, and decreased with less mature embryos. Somatic embryo response to mycorrhizal inoculation was dependent upon developmental stage, with embryos at later stages of maturation having highest survival, plantlet fonnation, and root colonization of roots by AM fungi. Inoculation with mycorrhizal fungus was effective only at later embryonic developmental stage, and only when roots were already present. We concluded that inoculation with AM fungi at the late stages of embryo development impraves survival and plantlet fonnation in vitro.
References ~Ial, B., Morei, c., and Gianinnazi-Pearson, V. 1990. Influence of vesicular-arbuscular mycorrhizal on phosphate fertilizer efficiency in the tropical planted with micropçropagated oil palm. Biol. Fertil. Soils, 9: 433-448. Giovanetti, M., and Mosse, B.V. 1980. An evaluation of techniques for measuring VA rnycorrhizal infection in roots. New Phytol. 84: 489-500.
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Gray, O.J., Conger, B.V., and Songstad, 0.0. 1987. Desiccated quiescent somatic embryos of orchardgrass for use as synthetic seeds. In Vitro Cell. Deve!. Biol. 23: 29-33. Hung, L.L.L.. O'Keefe, D., and Sylvia, O.M. 1991. Use of hydrogel as a sticking agent and carrier for vesicular-arbuscular mycorrhizal fungi. Mycol. Res. 95: 427-429. Molina, R. 1981. Ectornycorrhizal specificity in the genus Alnus. Cano I. BoI. 59: 325-334. Murashige, T., and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Physiol. Plant, 115: 473-497. Phillips, 1.M., ando Hayman, H.S. 1970. Improved procedure for clearing roots and staining parasitic and vesicular-arbuscular fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 55: 158-160. SAS Institute, Inc. 1985. SAS User's guide: Statistics. SAS Institute, Inc. Carry, N.C. Schultheis, I.R., Cantliffe, 0.1., Bryan, H.H., and Stofella, P.1. 1988. Planting methods to improve stand establishment, uniformity and earliness to flower in bell peper. 1. Am. Soe. Hortic. Sci. 99: 331-335. Strullu, 0.0., and Romand, C. 1987. Culture ascenique de vesicules isolees a partir d'endo Mycorrhizes et re-association in 'litro des racines de tomate. C. R. Acad. Sei, 305: 15-19. Taylor, K.c. 1986. The effect of hydrophilic polirner on media water retention and nutrient availability to Ligustrum leecidum, Hortic Sei. 21: 1159-ll61. White, P.R. 1963. A handbook of plant tissue culture. lacques Cottell Press, Lancaster, Pa.
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